Over 8 million people in the US suffer from peripheral arterial disease (PAD), which is characterized by narrowing of the arteries that supply blood flow to the limbs, leading to tissue ischemia. Biological approaches to enhance revascularization of the ischemic limb are promising. We previously demonstrated that endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) can improve blood perfusion in animals with induced hindlimb ischemia, an experimental model of PAD. However, poor cell survival limited their angiogenic potential. To address this limitation, in our parent R01 we developed aligned nanofibrillar collagen scaffolds that mimic the crimped (wavy) structure of native collagen fibrils that can enhance endothelial function and promote angiogenesis. In this sex differences administrative supplement, we seek to advance research related to women patients with PAD by incorporating sex as a key research outcome. The proposed studies intend to determine whether the sex of the donor iPSC-ECs or the sex of the recipient mice influences the angiogenic benefit of cell therapy in mouse model of PAD. Evaluating sex differences will enable a better understanding of how sex modulates the therapeutic efficacy of iPSC-ECs for treatment of PAD. Accordingly, in Specific Aim 1, we hypothesize that female iPSC-ECs will be more functional than male cells in the absence of estrogen treatment, but the function of both female and male cells improves in presence of estrogen treatment. To establish difference in endothelial function between men and women, we will derive iPSC-ECs from age-matched pairs of men and women with PAD. In the presence or absence of estrogen treatment, functional differences in iPSC-ECs derived from men and women will be performed using established assays to assess for angiogenic function, anti-inflammation, and nitric oxide production. We will also perform RNA sequencing for all chromosomes (including X and Y) to compare genetic differences among male and female cells with and without estrogen treatment. Specific Aim 2 will test the hypothesis that aligned nanofibrillar scaffolds will rescue the angiogenic function of male iPSC-ECs. Based on preliminary data that aligned nanofibrillar scaffolds enhance endothelial function and angiogenesis, male iPSC-ECs will be seeded on aligned or randomly oriented scaffolds in hypoxia (1% O2). In vitro quantitative comparison of cell viability, proliferation, metabolic activity, apoptosis, and nitric oxide production over the course of 14 days will be performed. To study the physiological effects of estrogen in modulating the function of iPSC-ECs after implantation into the ischemic hindlimb, the angiogenic effect of cell-seeded scaffold implantations in female recipient mice will be compared to that of male recipient mice. Output measures include laser Doppler spectroscopy for blood perfusion recovery, and confirmed by histological evaluation of capillary density. Together, these studies will reveal new insights into the contribution of sex differences to the efficacy of iPSC- ECs for treatment of PAD.